Expression of Matrix Metalloproteinase-2 (MMP-2) and Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) in Pancreatic Ductal Adenocarcinoma

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1 The Korean Journal of Pathology 2004; 38: 73-8 Expression of Matrix Metalloproteinase-2 (MMP-2) and Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) in Pancreatic Ductal Adenocarcinoma Mi Jin Gu Young Kyung Bae 1 Joon Hyuk Choi 1 Department of Pathology, Holy Trinity Hospital, 1 Yeungnam University College of Medicine, Daegu, Korea Received : December 5, 2003 Accepted : January 29, 2004 Corresponding Author Joon Hyuk Choi, M.D. Department of Pathology, Yeungnam University College of Medicine, Daemyung-dong, Nam-gu, Daegu , Korea Tel: Fax: jhcap@med.yu.ac.kr *Current affiliation of Mi Jin Gu is Department of Pathology, Fatima Hospital, Daegu, Korea. Background : Matrix metalloproteinase-2 (MMP-2) is known to be one of the key molecules for tumor invasion and metastasis. MMP-2 activity is modulated through interaction with the tissue inhibitor of metalloproteinase-2 (TIMP-2). The purpose of this study was to evaluate the expression of MMP-2 and TIMP-2 in pancreatic ductal adenocarcinoma. Methods : Using immunohistochemical staining, we investigated the expression of MMP-2 and TIMP-2 in 30 pancreatic ductal adenocarcinomas and 10 normal pancreas. Results : MMP-2 expression was present in tumor cells in 11 cases, and in stromal cells in 24 cases, out of 30 carcinomas. MMP-2 expression of tumor cells was significantly higher in poorly differentiated adenocarcinomas than in well/moderately differentiated adenocarcinomas, and in cases with vascular invasion than in cases without. MMP-2 expression was stronger in the marginal areas than in the central area of the tumor. TIMP-2 expression was detected in the tumor and stromal cells of all carcinomas. MMP-2 and TIMP-2 expression had no significant correlation with tumor size, lymph node metastasis, or TNM stage. MMP-2 expression was not correlated with TIMP-2 expression. Conclusions : These results suggest that MMP-2 expression may play an important role in the invasive property of pancreatic ductal adenocarcinoma, whereas TIMP-2 expression increases as a reaction to invasion. Key Words : Pancreas-Duct-Adenocarcinoma-MMP-2-TIMP-2 Pancreatic carcinoma is a disease with poor prognosis because it often is unresectable at diagnosis or it metastasizes to the liver in the early stages even after resection. 1-4 Several studies have investigated the molecular genetics of pancreatic carcinoma. However, the mechanism underlying the progression of pancreatic carcinoma is poorly understood, yet. Liotta et al. 5 reported that degradation of extracellular matrix (ECM), particularly basement membrane (BM), is mandatory for tumor invasion and metastasis. The current study demonstrated that proteinase, such as metalloproteinase, plays an important role in tumor invasion and metastasis. 6,7 Matrix metalloproteinases (MMP) are expressed in a variety tumor types including intestine, 8,9 breast, 10 stomach, 11,12 liver, 13 urinary bladder, 14 and esophagus. 15 The MMPs are a family of structurally related endopeptidases that degrades the macromolecular components of ECM and BM. 6,7,16,17 MMP-2 is 72-kilodalton and degrades type IV collagen and gelatin. 7,8,14,15 Therefore, MMP-2 is believed to be one of the key molecules for cancer invasion and metastasis. MMP-2 is secreted by human epithelial cells, fibroblasts, endothelial cells and macrophages as a latent form and is activated by loss of 80 amino acids from the amino terminus. 7,8 The activation of MMP-2 is regulated by the tissue inhibitor of metalloproteinase-2 (TIMP- 2). 1,3,6,8 TIMP is a 21-kilodalton, non-glycosylated protein that is secreted by many cells in fibroblasts, endothelial cells, vascular smooth muscle cells, and some tumor cells. 8,12 It is well known that TIMP can enhance ECM deposition and inhibit angiogenesis and MMP activity. In addition, TIMP regulates the growth of a variety of normal and transformed cell types. Under normal conditions, the balance between MMP and their corresponding inhibitors is a critical factor that maintains the homeostasis of connective tissue proteins. 18 The activated form of MMP-2 degrades BM, after which the activation of prommp-2 by membrane type MMPs is regarded as a key step in tumor invasion. TIMP-2 is expressed in many tumor types and forced expression of TIMP-2 will inhibit invasion in vivo and reduce metastasis formation in vivo. 18 Despite many reports of MMP action in human cancers, there are few findings about the role of MMP-2 and TIMP-2 for pancreatic carcinogenesis. To the best of our knowledge, a study on the MMP-2 and TIMP-2 expression of pancreatic ductal adenocarcinomas has not been reported in the Korean literature. The purpose of this study was to evaluate of role of MMP- 2 and TIMP-2 in tumor invasion and metastasis in pancreatic 73

2 74 Mi Jin Gu Young Kyung Bae Joon Hyuk Choi ductal adenocarcinoma. Materials MATATERIALS AND METHODS We selected 30 cases that had undergone surgery for pancreatic ductal adenocarcinoma and 10 cases of normal pancreas that had undergone operation for trauma or other organ tumors between 1986 and 2003, at Yeungnam University Hospital. Methods The specimens were fixed in 10% neutral formalin and embedded in paraffin. Immunohistochemical staining was performed using an avidin-biotin-peroxidase complex method. Deparaffinized sections were treated with 3% hydrogen peroxidase in methanol to block endogenous peroxidase. After antigen retrieval using an autoclave in citrate buffer solution, the specimens were allowed to react with a primary antibody for 90 min at room temperature. Primary antibodies were MMP-2 (mouse monoclonal MMP-2, 1:50, NeoMarkers, CA, USA) and TIMP-2 (mouse monoclonal TIMP-2, 1:50, NeoMarkers, CA, USA). An En Vision TM Chem Mate TM Detection Kit (DAKO Corporation, CA, USA) was used for the secondary antibody at room temperature for 30 min. After washing in Tris buffered saline for 10 min, 3,3 - diaminobenzidine (DAB) was used as a chromogen, and then Mayer s hematoxylin counterstain was applied. Immunohistochemical evaluation In the normal pancreas, the results was regarded as positive when more than 10% of epithelial cells and stromal cells showed positive immunostaining. The percentage of tumor cells in each section exhibiting positive immunohistostaining was estimated by counting 500 cells. MMP-2 and TIMP-2 expression was classified into three categorizes depending on the percentage of stained cells: -; 0 to 10% positive tumor cells, +1; 10 to 50% positive tumor cells, and +2; more than 50% of positive tumor cells. Statistical analyses Testing for the association between the MMP-2 and TIMP-2 and histopathologic parameters was performed by 2 analysis. The unpaired Student s t-test was used to test for statistical differences between groups, with p-values of less than 0.05 being considered statistically significant. RESULTS Clinical and pathologic findings The median age of the patients, 17 males and 13 females, was 56.5 years (range 32 to 76). The mean tumor size was 3.82 cm (range 2.0 to 6.5). The locations of the pancreatic ductal adenocarcinomas were 17 cases in the head, 4 in the body, 4 in the tail, 2 in the head and body, and 3 in the body and tail. Histologic examination revealed well differentiation in 11 cases, moderate in 5, and poor in 14. Twenty-four cases revealed vascular invasion, and 16 neural invasion. According to tumor-node-metastasis (TNM) classification (AJCC, sixth edition), 16 cases were stage IIa, 12 stage IIb, one each stages III and IV, and zero stage I. Immunohistochemical findings of MMP-2 In the normal pancreas, MMP-2 was not expressed in ductal, islet, or acinar cells, but was faintly expressed in stromal cells in all cases (Table 1) (Fig. 1). In the pancreatic ductal adenocarcinomas, MMP-2 expression was present in the cellular membrane of tumor cells with or without cytoplasmic staining in 11/30 (36.7%) cases and in stromal cells in 24/30 (80.0%) cases (Fig. 2) (Table 1). MMP-2 expression of tumor cells was significantly higher in poorly differentiated adenocarcinomas than in well/moderately differentiated adenocarcinomas (p<0.05). There was no significant correlation between MMP-2 expression and other clinicopathologic parameters such as tumor size, lymph node metastasis, and TNM stages (Table 2). MMP-2 expression was significantly higher in cases with vascular invasion than in cases without (p<0.05) (Table 2). MMP-2 expression was stronger in tumor cells in the marginal areas of the tumor than in the central area of the tumor. There was no significant correlation between tumor cells and stromal cells on MMP-2 expression. Immunohistochemical findings of TIMP-2 In the normal pancreas, TIMP-2 was expressed in the ductal, islet, acinar and stromal cells in all cases (Table 1) (Fig. 3). In the pancreatic ductal adenocarcinomas, TIMP-2 expression was detected in the cellular membrane of tumor cells with or

3 MMP-2 and TIMP-2 Expression in Pancreatic Ductal Adenocarcinoma 75 Fig. 1. Expression of marix metalloproteinase-2 (MMP-2) is detected in the stromal cells but not in the ductal or acinar cells of the normal pancreas by immunohistochemistry. Fig. 2. Expression of marix metalloproteinase-2 (MMP-2) is found in the cytoplasm, of prominently in the tumor cells and faintly in the stromal cells by immunohistochemistry. Fig. 3. Expression of tissue inhibitor of metalloproteinase-2 (TIMP- 2) is detected in the cytoplasm of normal acinar, ductal cells, and stromal cells by immunohistochemistry. without cytoplasmic staining and in the stromal cells of all 30 cases (Fig. 4). The staining in the stromal cells of the normal pancreas and tumor tissue was intensely positive. TIMP-2 expression was stronger in the pancreatic ductal adenocarcinomas than in the normal ductal cells. There was no correlation between TIMP-2 expression and tumor size, histologic grade, vascular Fig. 4. Expression of tissue inhibitor of metalloproteinase-2 (TIMP-2) is found in the cytoplasm of tumor cells and stromal cells by immunohistochemistry. invasion, lymph node metastasis, or TNM stages. Relationship of MMP-2 and TIMP-2 expression in tumor cells and tumor stromal cells There was no significant correlation between MMP-2 and

4 76 Mi Jin Gu Young Kyung Bae Joon Hyuk Choi Table 1. The expression of MMP-2 and TIMP-2 in normal pancreas and pancreatic ductal adenocarcinoma Table 3. Relationship of MMP-2 and TIMP-2 expression of tumor cells of pancreatic ductal adenocarcinoma Tissue MMP-2 positive Epithelial cell Stromal cell TIMP-2 positive Epithelial cell Stromal cell Parameters TIMP-2 <10% (-) 10-50% (+1) >50% (+2) p value Normal 0/10 (0%) 10/10 (100%) 10/10 (100%) 10/10 (100%) pancreas faintly + ductal, acinar, (n=10) islet cells Ductal adeno-11/30 (36.7%) 24/30 (80.0%) 30/30 (100%) 30/30 (100%) carcinoma (n=30) Table 2. Relationship between MMP-2 expression and clinical parameters in tumor and stromal cells of pancreatic duct adenocarcinoma Parameters No. of cases Positive cases Tumor cells (%) p value TIMP-2 expression in either tumor cells or tumor stromal cells (Table 3, 4). DISCUSSION Positive cases Stromal cells (%) p value Size <4 cm 15 6 (40.0) N-S 13 (86.7) N-S 4-6 cm 14 5 (35.7) 10 (71.4) >6 cm 1 0 (0) 1 (100) Differentiation Well 11 3 (27.3) p< (90.9) N-S Moderate 14 4 (28.6) 10 (71.4) Poor 5 4 (80.0) 4 (80.0) Vascular invasion Positive (45.8) p< (75.0) N-S Negative 6 0 (0) 6 (100) Neural invasion Positive 16 5 (31.3) N-S 13 (81.3) N-S Negative 14 6 (42.9) 11 (78.6) Lymph node metastasis Positive 12 4 (33.3) N-S 10 (83.3) N-S Negative 18 7 (38.9) 13 (72.2) Stage I 0 0 (0) N-S 0 (0) N-S II 28 4 (14.3) 23 (82.1) III 1 1 (100) 0 (0) IV 1 0 (0) 1 (100) N-S, not significant. Ductal adenocarcinoma of the exocrine pancreas comprises about 85% of all cases of pancreatic malignancy and is characterized by local invasion of adjacent structures, perineural invasion, early metastasis to the lymph node and liver, and strong desmoplastic stromal reaction. Because of unresponsiveness to chemotherapy and radiotherapy, low resectability rates after diagnosis, MMP-2 <10% (-) 0 (0%) 7 (36.8%) 12 (63.2%) N-S 10-50% (+1) 0 (0%) 1 (25.0%) 3 (75.0%) >50% (+2) 0 (0%) 1 (14.3%) 6 (85.7%) N-S, not significant. Table 4. Relationship of MMP-2 and TIMP-2 expression of stromal cells of pancreatic ductal adenocarcinoma Parameters TIMP-2 <10% (-) 10-50% (+1) >50% (+2) p value MMP-2 <10% (-) 0 (0%) 6 (100%) 0 (0%) N-S 10-50% (+1) 0 (0%) 17 (81.0%) 4 (19.0%) >50% (+2) 0 (0%) 2 (66.7%) 1 (33.3%) N-S, not significant. early recurrence after resection, and extremely poor survival rates, the 5-year survival rate has been reported at less than 0.4%. 1,3,4,19,20 MMP has been reported to play a key role in the sequence of events that leads to tumor invasion and metastasis. Several studies about MMP-2 expression in pancreatic adenocarcinoma have been described. Nagakawa et al. 19 reported that MMP-2 was expressed in 40.6% of tumor cells of pancreatic ductal adenocarcinoma by immunohistochemistry. Meanwhile, our study showed 36.7% MMP-2 expression in pancreatic tumor cells and 80.0% in stromal cells. Koshiba et al. 16 reported that the MMP-2 activation ratio in pancreatic carcinoma was significantly higher than that of chronic pancreatitis and normal pancreatic tissue by zymography. Therefore, activated MMP may be closely associated with pancreatic cancer. In our study, MMP-2 was only faintly expressed in normal pancreatic stromal cells. Davies et al. 21 described that MMP-2 was correlated with clinical stage in tumors of the lung, breast, and urinary bladder. Toshiba et al. 2 reported that the MMP-2 activation ratio in PT3 tumors was significantly higher than that in PT1 tumors. In our study, there was no significant difference between the MMP-2 expression and clinical stages, because of the single cases of stages III and IV and the absence of any stage I. Gong et al. 6 reported that a higher level of MMP and a lower level of TIMP-1 gene expression were observed in the cases of pancreatic ductal carcinoma with poor differentiation than in the cases with well differentiation. In this study, MMP- 2 expression of tumor cells was significantly higher in the poorly differentiated adenocarcinomas than in the well/moderately differentiated adenocarcinomas. However, an inverse relationship between differentiation and MMP expression in lung, 22 colon 8

5 MMP-2 and TIMP-2 Expression in Pancreatic Ductal Adenocarcinoma 77 and gallbladder 23 cancers was described. Nagakawa et al. 19 reported that expression of MMP-2 in pancreatic cancer was strongly related to the vascular wall destruction by cancer. Kuniyasu et al. 24 reported especially high levels of MMP-2 and MMP-9 in recurrent pancreatic tumor margins. In our study, MMP-2 was highly expressed in cases with vascular invasion and in tumor cells of marginal areas compared to those of the central area. These results suggest that expression of MMP-2 contributes more to tumor invasion and metastasis than the proliferation of tumor cells. Ellenrieder et al. 7 reported that expression and activation of MMP-2 were strongly associated with the extent of the desmoplastic reaction in pancreatic tissue. Hoyhtya et al. 25 described that TIMP-2 was localized in most of the cases in the stroma surrounding the tumor cells in endometrial, ovarian and prostatic carcinomas. In our study, TIMP-2 was expressed in not only normal pancreatic tissue, but also in the pancreatic ductal adenocarcinoma tumor cells and stromal cells. However, TIMP-2 expression intensity was stronger in the pancreatic ductal adenocarcinomas than in the normal pancreas. In our study, TIMP-2 was highly expressed in tumor and stromal cells, compared to that reported in previous studies with other organ tumors. 6,23,26 This increase may be associated with stromal response to tumor invasion with activated MMP-2 and/or dense fibrotic reaction that prevents tumor cells from invading adjacent tissue and limits tumor growth. Yamamoto et al. 3 reported no correlation between TIMP expression and tumor size, differentiation, and stages. There was no significant correlation between TIMP-2 expression and clinical parameters in our study. Bramhall et al. 1 suggested that reduced expression of TIMP-2 could contribute to the aggressive phenotype and desmoplastic response. However, a more recent work suggested that TIMPs were multifunctional molecules, with apparent paradoxical effects on tumor progression. 26 TIMP-1 and TIMP-2 are both known to have not only growth-factor-like properties but also MMP inhibitory functions. Ree et al. 27 reported with that TIMP-2 mrna levels were significantly higher in breast cancers from patients who developed distant metastasis than in those who remained free of metastatic disease. Furthermore, Visscher et al. 28 suggested that clinical outcome is more closely related to elevated TIMP-2 expression than to the corresponding MMPs. The inversion and co-ordinate expression between MMP-2 and TIMP-2 have been reported in many different tumors. 6,22,23,26 In our study, there was no significant correlation between MMP-2 and TIMP-2 expression in tumor cells and stromal cells. Nagase 29 reported that TIMP-2 could inhibit MMP-2 activity at high concentrations and activate MMP-2 activity at low levels. Matsuyama et al. 30 reported that TIMP can immediately suppress MMP-2 activation, however, TIMP cannot restrain MMP-2 activation when other MMPs are present. The elevated TIMP-2 expression of tumor cells in our study may have been associated with activated MMP-2. Therefore, whereas MMP-2 expression may play an important role in the tumor invasion of pancreatic ductal adenocarcinomas, TIMP-2 expression may be associated with the response to tumor invasion. Our study had a statistical limitation of uneven selected parameters. Therefore, additional studies are required to complement our data. REFERENCES 1. Bramhall SR, Neoptolemos JP, Stamp GWH, Lemoine NR. Imbalance of expression of matrix metalloproteinase (MMPs) and tissue inhibitors of the matrix metalloproteinases (TIMPs) in human pancreatic carcinoma. J Pathol 1997; 182: Koshiba T, Hosotani R, Wada M, et al. Involvement of matrix metalloproteinase-2 activity in invasion and metastasis of pancreatic carcinoma. 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